Usutu virus (USUV, Flaviviridae) was first reported in Europe in Austria in 2001, where it caused wild bird (mainly blackbird) mortality until 2005. Since 2006 no further USUV cases were diagnosed in the country. However, the virus emerged in other European countries (Hungary, Italy, Switzerland, Spain, Germany and the Czech Republic) between 2005 and 2011. In 2016, widespread USUV-associated wild bird mortality was observed in Germany, France, Belgium and the Netherlands. In this study, we report the results of passive monitoring for USUV in Austria and Hungary between 2010 and 2016. In Hungary, USUV caused sporadic cases of wild bird mortality between 2010 and 2015 (altogether 18 diagnosed cases), whereas in summer and autumn 2016 the number of cases considerably increased to 12 (ten blackbirds, one Eurasian jay and one starling). In Austria, USUV was identified in two blackbirds in 2016. Phylogenetic analyses of coding-complete genomes and partial regions of the NS5 protein gene revealed that USUVs from Hungary between 2010 and 2015 are closely related to the virus that emerged in Austria in 2001 and in Hungary in 2005, while one Hungarian sequence from 2015 and all sequences from Hungary and Austria from 2016 clustered together with USUV sequences reported from Italy between 2009 and 2010. The results of the study indicate continuous USUV circulation in the region and exchange of USUV strains between Italy, Austria and Hungary.
Generally regarded as extracellular pathogens, molecular mechanisms of mycoplasma persistence, chronicity and disease spread are largely unknown. Mycoplasma agalactiae, an economically important pathogen of small ruminants, causes chronic infections that are difficult to eradicate. Animals continue to shed the agent for several months and even years after the initial infection, in spite of long antibiotic treatment. However, little is known about the strategies that M. agalactiae employs to survive and spread within an immunocompetent host to cause chronic disease. Here, we demonstrate for the first time its ability to invade cultured human (HeLa) and ruminant (BEND and BLF) host cells. Presence of intracellular mycoplasmas is clearly substantiated using differential immunofluorescence technique and quantitative gentamicin invasion assays. Internalized M. agalactiae could survive and exit the cells in a viable state to repopulate the extracellular environment after complete removal of extracellular bacteria with gentamicin. Furthermore, an experimental sheep intramammary infection was carried out to evaluate its systemic spread to organs and host niches distant from the site of initial infection. Positive results obtained via PCR, culture and immunohistochemistry, especially the latter depicting the presence of M. agalactiae in the cytoplasm of mammary duct epithelium and macrophages, clearly provide the first formal proof of M. agalactiae's capability to translocate across the mammary epithelium and systemically disseminate to distant inner organs. Altogether, the findings of these in vitro and in vivo studies indicate that M. agalactiae is capable of entering host cells and this might be the strategy that it employs at a population level to ward off the host immune response and antibiotic action, and to disseminate to new and safer niches to later egress and once again proliferate upon the return of favorable conditions to cause persistent chronic infections.
BackgroundIn spring 2015, an outbreak of porcine reproductive and respiratory syndrome (PRRS) struck Lower Austria caused by a PRRS virus (PRRSV) strain spreading rapidly among both previously PRRSV negative and vaccinated pig herds. This case report describes the first well-documented emergence of the PRRSV strain responsible for this outbreak.Case presentationA PRRSV seronegative piglet-producing farm in Lower Austria encountered losses in foetuses and suckling piglets of up to 90 %; clinical signs in sows and nursery piglets included fever and reduced feed intake. Additionally, high percentages of repeat breeders and losses of up to 40 % in nursery piglets occurred. An infection with PRRSV was suggested by the detection of antibodies by enzyme linked immunosorbent assay and confirmed by quantitative real time PCR. The underlying PRRSV strain, termed AUT15-33, was isolated by passage on porcine alveolar macrophages, partially sequenced (ORF2-7) and grouped as PRRSV-1, subtype 1. In phylogenetic analysis of the genome region coding for the structural proteins, ORF2-7, AUT15-33 clustered with Belgian strains but identities were as low as 88 %. In contrast, analysis of ORF7 sequences revealed a close relationship to Croatian strains from 2012 with an identity of 94 – 95 %.ConclusionsIn the year following the outbreak, the same PRRSV strain was identified repeatedly in different regions of Austria. It can be speculated that the new strain has novel advantageous properties.
Bacillus cereus is a gram-positive pathogen mainly known to evoke two types of foodborne poisonings. The diarrheal syndrome is caused by enterotoxins produced during growth in the intestine. In contrast, the emetic type is caused by the dodecadepsipeptide cereulide pre-formed in food. Usually, both diseases are self-limiting but occasionally more severe forms, including fatal ones, are reported. Since the mechanisms of cereulide toxin uptake and translocation within the body as well as the mechanism of its toxic action are still unknown, we used a porcine model to investigate the uptake, routes of excretion and distribution of cereulide within the host. Pigs were orally challenged with cereulide using single doses of 10–150 μg cereulide kg-1 body weight to study acute effects or using daily doses of 10 μg cereulide kg-1 body weight administered for 7 days to investigate effects of longtime, chronic exposure. Our study showed that part of cereulide ingested with food is rapidly excreted with feces while part of the cereulide toxin is absorbed, passes through membranes and is distributed within the body. Results from the chronic trial indicate bioaccumulation of cereulide in certain tissues and organs, such as kidney, liver, muscles and fat tissues. Beside its detection in various tissues and organs, our study also demonstrated that cereulide is able to cross the blood–brain–barrier, which may partially explain the cerebral effects reported from human intoxication cases. The neurobehavioral symptoms, such as seizures and lethargy, observed in our porcine model resemble those reported from human food borne intoxications. The rapid onset of these symptoms indicates direct effects of cereulide on the central nervous system (CNS), which warrant further research. The porcine model presented here might be useful to study the specific neurobiological effect in detail. Furthermore, our study revealed that typical diagnostic specimens used in human medicine, such as blood samples and urine, are not suitable for diagnostics of food borne cereulide intoxications. Instead, screening of fecal samples by SIDA-LC-MS may represent a simple and non-invasive method for detection of cereulide intoxications in clinical settings as well as in foodborne outbreak situations.
Usutu virus (USUV), a mosquito‐borne flavivirus closely related to West Nile virus, emerged in Austria in 2001, when it caused a considerable mass‐mortality of Eurasian blackbirds. Cases in birds increased until 2003 and quickly declined thereafter, presumably due to developing herd immunity. Since 2006, no further cases were recorded, until two blackbirds were tested positive in 2016. In Hungary, USUV first appeared in 2005 and has caused only sporadic infections since then. Initially, the only genetic USUV lineage found across both countries was Europe 1. This changed in 2015/2016, when Europe 2 emerged, which has since then become the prevalent lineage. Due to dispersal of these strains and introduction of new genetic lineages, USUV infections are now widespread across Europe. In 2009, the first cases of USUV‐related encephalitis were described in humans, and the virus has been frequently detected in blood donations since 2016. To monitor USUV infections among the Austrian wild bird population in 2017/2018, 86 samples were investigated by RT‐PCR. In 67 of them, USUV nucleic acid was detected (17 in 2017, 50 in 2018). The majority of succumbed birds were blackbirds, found in Vienna and Lower Austria. However, the virus also spread westwards to Upper Austria and southwards to Styria and Carinthia. In Hungary, 253 wild birds were examined, but only six of them were infected with USUV (five in 2017, one in 2018). Thus, in contrast to the considerable increase in USUV‐associated bird mortality in Austria, the number of infections in Hungary declined after a peak in 2016. Except for one case of USUV lineage Africa 3 in Austria in 2017, Europe 2 remains the most prevalent genetic lineage in both countries. Since USUV transmission largely depends on temperature, which affects vector populations, climate change may cause more frequent USUV outbreaks in the future.
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